1. Field of the Invention
The present invention relates to a method for the rapid and accurate detection and quantification of malaria-causing protozoans in the genus Plasmodium using mass spectrometry analysis of red blood cells. The invention is based on the discovery that unbound heme that is concentrated in malaria parasites, can be detected in a blood sample and quantified using mass spectrometry. The method can also be applied to detect and quantitate other heme-concentrating parasites including those belonging to the phylum Apicomplexa, and to detect any other heme-concentrating microorganism, including bacteria.
2. Description of the Related Art
Malaria is a disease affecting man and other animals, caused by different species of the parasite Plasmodia that are transmitted by mosquito bites. During a complex life cycle involving insect and animal hosts, the malaria parasite invades and destroys erythrocytes. Despite intense efforts to combat it, malaria still afflicts more than 500 million people. Each year, malaria infections kill between one and two million people in 103 countries, predominantly children. The emergence of drug-resistant strains and slow progress in developing an effective vaccine has compounded efforts to control the spread of the disease. Malaria in humans is caused by four different species of protozoans in the genus Plasmodium: P. falciparum, P. vivax, P. malariae and P. ovale, with P. falciparum being the most lethal.
The Plasmodium life cycle proceeds through several asexual and sexual stages. Plasmodium sporozoites, transmitted by female Anopheles mosquitoes, are injected into the blood of an animal host together with mosquito saliva. After initial proliferation in the liver, parasites in the merozoite stage are released back into the blood stream. A single merozoite then invades a red blood cell (RBC) and matures by forming a ring-shaped cell. In about 24 hours the matured parasite enters the trophozoite stage, during which most of the RBC cytoplasm, including hemoglobin, is catabolized. Through the final (schizont) stage in the RBC, the parasite undergoes several divisions to produce up to thirty-two new merozoites that burst the host RBC and invade new erythrocytes.
Rapid, sensitive and reliable methods for malaria detection are a factor that determines the ultimate success in controlling, restricting and eradicating this disease. Moreover, accurate parasitemia quantification is indispensable in malaria treatment, in screening new drug and candidate vaccine efficacy, and in identifying the emergence of drug-resistant parasite strains. While optical microscopy of Giemsa-stained blood smears is still considered the “gold standard” for malaria detection, it is a time consuming process requiring about one half hour per diagnosis using experienced technicians. Several new diagnostic techniques have been developed in recent years including fluorescence microscopy, PCR-based assays, serological (“dipstick”) antigen detection, and flow cytometry (with or without laser light depolarization monitoring). However, currently available techniques are either too time-consuming, have low sensitivity or specificity, require trained technicians, provide poor quantitation of parasitemia, or are too expensive for mass screening. Based on the foregoing there is a clear need for a method to diagnose malaria and quantify the level of malaria parasites in an infected animal, that does not suffer the deficiencies of the prior art.
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4. Definitions
Bound heme means a molecule that is non-covalently bound to a polypeptide chain, e.g., one heme bound to each of the four subunits of hemoglobin (the oxygen-binding hemoglobin prosthetic group).
Heme (ferriprotoporphyrin IX, C34H32N4O4Fe) consists of a particular planar tetrapyrrole ring system that chelates iron.
Heme-concentrating parasite means any parasite including but not limited to Plasmodium that concentrates unbound heme including: parasites of the phylum Plathyhelminthes (the flatworms), Order Strigeiformes, family Schistosomatidae, species Schistosoma (blood flukes causing shistosomiasis); parasites of the phylum Apicomplexa; and any other parasites that concentrates heme.
Malaria parasite means any species of Plasmodium that causes malaria.
Parasitemia means the number of parasites per unit of blood.
Unbound heme means free heme molecules, including heme molecules that are part of the malaria pigment hemozoin, e.g. heme that is not bound to each of the four subunits of hemoglobin or other protein.